Costunolide, a sesquiterpene lactone isolated from the root of Saussurea lappa Clarke, is known to have a variety of biological activities, including anti-carcinogenic and anti-fungal activities. Here, we demonstrated the inhibitory effect of costunolide on the protein and mRNA expression of interleukin-1β (IL-1β) in LPS-stimulated RAW 264.7 cells. We also showed that costunolide suppressed the transcriptional activity of the IL-1β promoter. Moreover, costunolide inhibited the activity of AP-1 transcription factor, and the phosphorylation of MAPKs, including SAPK/JNK and p38 MAP kinase. The inhibitory effect of costunolide on AP-1 activity was also confirmed by an electrophoretic mobility shift assay. Additionally, specific inhibitors of SAPK/JNK and p38 MAP kinase, SP600125 and SB203580, also suppressed LPS-induced increase in IL-1β gene expression and AP-1 DNA binding. Taken together, these results demonstrate that costunolide inhibits IL-1β gene expression by blocking the activation of MAPKs and DNA binding of AP-1 in LPS-stimulated RAW 264.7 cells.